Sex: male age (yrs): 50 crt: Capox ctn: T4N0 surg: APR trg: 4 downst: NO downs: NO resp: NO cpr: NO
Treatment protocol
The study included a total of 35 consecutive patients with locally advanced rectal cancer treated at Virgen de las Nieves Hospital from 2008 until 2010. Written informed consent was obtained from all the patients, and the study protocol was approved by the local Clinical Research (Ethics) Committee. They were distributed in two subgroups following a histological classification of response to treatment. Pretherapeutic staging was performed, including complete medical history and physical evaluation, digital rectal examination, endorectal ultrasound, rigid rectoscopy, colonoscopy, PET/TC and chest x-ray. Tumor samples were prospectively obtained during the rectoscopy. All patients subsequently received a total dose of 50.4Gy of radiatio (28 fractions of 1.8Gy) associated with capecitabine or capecitabine and oxaliplatine. Standardized surgery was performed, including total mesorectal excision, after an interval of 8 weeks after chemoradiotherapy. Tumor response was assessed in surgical specimen by semi-quantitative pathological examination (regression grade) including UICC stage. Histopathological tumor regression was graded based on the semiquantitative 5 point tumor regression grading (TRG) system: TRG1 or a TRG2 scores were considered Responders, whereas TRG3, TRG4, and TRG5 scores were classified as Non-Responders. In addition, the tumor regression or radiotherapy effects were assessed by positron emission tomography (18F-FDG PET). Tumor samples of LARC were collected from 35 patients. Nine patients were finally excluded due to the poor quality of the RNA or contradictory results of Mandard´s criteria and histopathological downstaging.
Extracted molecule
total RNA
Extraction protocol
Frozen sample materials were provided by the Tissue and Tumor Bank, Department of Pathology at the Virgen de las Nieves Hospital. RNAs were extracted from macrodissected frozen samples according to standard procedures using Rnesasy minikit (Qiagen Sciences). Quantity and integrity of RNAs were checked by spectrophotometry in a NanoDropTM (ND-1000, DE, USA) and in an ExperionTM automated electrophoresis system (Bio-Rad, Richmond, VI, USA), respectively. Prior to extraction, 8 µm section were stained with hematoxylin and eosin for microscopic examination to check the percentage of tumor cells. The percentage of tumor cells was estimated by an experienced pathologist in each case via visual inspection.
Label
Cy5
Label protocol
reverse transcription cRNAs were labeled with Cy5 Streptavidine (Amersham Biosciences, Sweden)
Hybridization protocol
Hybridization of whole genome human genes included in CodeLink bioarrays (Applied Microarrays, Tempe, AZ, USA) was performed overnight at 37°C in a shaker incubator (Innova 4080, New Brunswick®, NJ, U.S.A.). The hybridization reactions were done in duplicate.
Scan protocol
Microrrays were read with a GenePix 4000B laser scanner (Axon Instruments, CA, USA)
Microrrays were read, quantified and normalized using CodeLink Software 5.0 (Applied Microarrays, Tempe, AZ, USA). Only probes of type ’DISCOVERY’ were taken into account. Negative values for signal intensities were set to 0,0. Probes with missing values for any array were disregarded. Probes were identified by the Codelink Probe IDs (GE-XXXX). For patients with 2 replicates, average values were computed.
